The following application is to continue support an inter-institutional Program of Excellence in Gene Therapy (PEGT). The participating institutions include: Stanford University, The Children's Hospital of Philadelphia, and The University of Pennsylvania and investigators with a well-documented history of collaboration including the successful implementation of an AAV- liver based gene therapy trial. We have used the information learned from this trial to develop new preclinical clinical trials. In this application, there are 3 preclinical gene therapy proposals 2 clinical trials. There are three "local" core proposals (for the investigators of this PEGT), a Research Grade AAV, Administrative, and Clinical Core. We also propose a unique training program to offer post-doctoral candidates research training in gene therapy. A National Morphology core is designed to provide all NHLBI investigators with unique services to further advance the study of gene transfer and/or transgene expression in animal models. The common theme of this proposal is AAV-mediated gene based therapies for hemophilia: (#1) Dr. Katherine High will test the hypothesis that the bleeding diathesis in hemophilia patients with inhibitory antibodies can be treated with a gene therapy approach. This grant builds on a decade of clinical experience with recombinant Vila and on the investigator's expertise with coagulation proteins and their expression using AAV vectors. (#2) Dr. Haig Kazazian and colleagues will build on their success in treating hemophilia A mice using new AAV serotypes. They will determine optimal conditions for liver-based gene transfer in fetal monkeys, and validate these conditions in neonatal monkeys and neonatal hemophilia A dogs. (#3) Dr. Mark Kay plans to develop novel AAV pseudotyped vectors that may evade immunological parameters that have limited efficacy in human trials. His group will use non-mammalian primate AAVs and snuffled capsid libraries to make novel vectors and test them in the appropriate animal models. (#4) Dr. Catherine Manno will continue an AAV-2 liver based trial using transient immunosuppressive therapy to attempt to eliminate a T cell mediated elimination of transduced hepatocytes in humans. (#5) Dr. Bertil Glader is proposing a clinical trial using AAV-8 vectors that based on preclinical studies suggesting that this vector pseudotype has advantages over AAV-2. Nonetheless, as has been learned during the previous funding period, even the best animal models and preclinical studies cannot always predict the outcome in humans, supporting the need for multiple approaches. The combined efforts of this group of investigators have a long and productive history that will further research towards a cure for hemophilia. INDIVIDUAL PROJECTS AND CORE UNITS: Project 1: Novel Therapeutic Approach for Hemophilia Using Engineered Secreted FVIIa. (High, Katherine) DESCRIPTION (provided by applicant): The goal of Project 1 is to determine the feasibility of expression of FVIIa as the transgene in a gene therapy approach for hemophilia. Work with recombinant proteins has established that hemophilia, caused by mutations in genes encoding Factors VIII or IX in the intrinsic pathway of coagulation, can be treated by infusion of activated Factor VII, an enzyme of the extrinsic pathway. In the previous funding period, we created an engineered FVIIa variant, expressed it in a recombinant AAV vector, introduced the vector into the livers of hemophilic mice, and demonstrated long-term expression of activated FVII and amelioration of the hemophilic phenotype. We propose to build on this proof-of-concept by 1) defining precisely the minimum level of VIla required for improvement in hemostasis, and the maximum safe tolerated levels. This will be accomplished by studying hemostatic endpoints in both vector-treated and transgenic Vila-expressing mice. We will use state-of-the-art methods to examine kinetics of clot formation, clot stability, and clot composition as a function of circulating levels of FVIIa in hemophilic mice. 2) In the second aim, we will extend this work to the large animal model of hemophilia. In these experiments we will infuse an AAV vector expressing activated canine FVII into the liver of dogs with severe hemophilia, and determine levels that are safely tolerated and that result in improved hemostasis. 3) Finally, we will capitalize on a novel vector delivery method that we have developed during the previous funding period. This route of administration exploits the extensive capillary network of skeletal muscle to effect transduction of large areas of skeletal muscle. We will determine whether regional intravascular delivery allows us to achieve adequate levels of FVIIa expression using a target organ skeletal muscle) that is accessible for nearly all hemophilia patients, even those with severe liver changes due to viral hepatitis. Successful development of a Vila-based gene transfer strategy would be applicable for both -VIII and FIX deficiency, would avoid problems of immune response to the transgene product (FVIII or FIX) identified in preclinical gene transfer studies, by using a transgene to which the recipient is fully tolerant, and would circumvent problems of short half-life and need for IV infusion that characterize therapy with the recombinant Vila protein. (End of Abstract)